Amino acids are important food additives in both human and animal diets and their production and purification have become vital to numerous food industries. Many amino acids are made either chemically or through fermentation processes which require the separation and isolation of the desired amino acids from the broth.
Many amino acids exist as two optically active enantiomers, the L and D-isomers. It is often preferred in many applications to separate the two from their racemic mixture that is produced during chemical processing. L-phenylalanine for example, is a component of the popular dipeptide sweetener aspartame, technically known a alpha-L-aspartyl-L-phenylalanine methyl ester (APM). It is also known that when the dipeptide is comprised of the two L-isomers of aspartic acid and phenylalanine, it is sweet, whereas their D-L, L-D, D-D etc. enantiomers are not. Moreover, any of the enantiomers which contain D-phenylalanine are worthless as sweeteners.
Aspartame can by synthesized by any one of several biochemical processes but these generally involve a coupling reaction whereby aspartic acid is joined with either phenylalanine or its methyl ester. Hence, the finished product must be separated from its enantiomers and any unreacted phenylalanine and aspartic acid. It is economically advantageous to conserve L-phenylalanine by hydrolysing the non-sweet esters and recovering the phenylalanine. However, during the various chemical process steps used to make APM and recover the leftover phenylalanine, some of the L-phenylalanine is racemized. Therefore, when this phenylalanine is recovered, it contains some of the D-isomer. Since the D-isomer cannot be utilized in the manufacture of the dipeptide sweetener, it would be useful to be able to separate the L-phenylalanine isomer as economically as possible from the racemic mixture.
It is an object of the present invention to crystallize and isolate pure L-isomers of an amino acid from a racemic mixture when the racemate has more of the L-isomer than the D-form. More specifically, it is an object of the present invention to obtain pure L-phenylalanine from a racemic mixture comprised of both the L- and D-forms.
Various methods for separating the L and D isomers, are known and generically are referred to as resolution. The most common method of resolving D,L-mixtures involves combining them with an optically active compound known as a resolving agent, followed by fractional crystallization of the resulting mixture of compounds (diastereroisomers) in solution. For practical resolution, it is necessary to find a combination of resolving agent and solvent which will give good crystallization behavior together with a pronounced difference in solubility between the diastereoismers. Examples of this technique as applied to the resolution of an amino acid are in U.S. Pat. Nos. 2,556,907, Emmick, R., and 2,657,230, Rogers, A. These patents discuss methods to resolve D, L-lysine in which optically active glutamic acid is employed as the resolving agent.
More recently, phenylalanine has been resolved by enzymatic hydrolysis of its diastereroisomers. The enzyme, chymotrypsin, selectively hydrolyzes L-phenylalanine esters. Hence L-phenylalanine is recovered from a mixture of the D,L-phenylalanine ester. An example of this process is Eur. Pat. Appl. No. EP 174,862, Empie, M., (8/17/84).
However, the aforementioned methods of the prior art require an additional step beyond that of the fermentation involving the use of a resolving agent in a chemical or enzymatic reaction or some combination of these to form a derivative of phenylalanine in order to isolate the desired isomer. The present invention permits the separation of the L-isomer from the racemic mixture and in the same step, its purification from other amino acids, salts, etc., without the requirement of a resolving agent, the subsequent formation of a derivative of the amino acid in question and without enzymatic reaction.